NO346530B1 - Applications of pegylated interleukin-10 (PEG-IL-10) to prevent metastases of cancer or tumor in the lungs. - Google Patents

Description

Field of the invention

The present invention concerns applications of pegylated interleukin-10 (PEG-IL-10) to prevent metastases of cancer or tumors in the lungs.

The background of the invention

Cancers and tumors can be controlled or removed by the immune system. The immune system includes several types of lymphoid and myeloid cells, e.g. monocytes, macrophages, dendritic cells (DC), eosinophil cells, T cells, B cells and neutrophil cells. These lymphoid and myeloid cells form secreted signaling proteins called cytokines. The cytokines include interleukin-10 (IL-10), interferon-gamma (IFNy), IL-12 and IL-23. The immune response includes inflammation, i.e. the accumulation of immune cells systemically or in a given part of the body. In response to an infectious agent or a foreign compound, the immune cells secrete cytokines, which in turn modulate the proliferation, development, differentiation or migration of the immune cells. An excessive immune response can lead to pathological consequences, e.g. autoimmune disorders, while a weakened immune response can lead to cancer. The immune system's antitumor response includes innate immunity, e.g. mediated by macrophages, NK cells and neutrophil cells, and adaptive immunity, e.g. mediated by antigen-presenting cells (APC), T cells, and B cells (see, e.g., Abbas, et al. (eds.) (2000) Cellular and Molecular Immunology, W.B. Saunders Co., Philadelphia, PA; Oppenheim and Feldmann (ed.) (2001) Cytokine Reference, Academic Press, San Diego, CA; von Andrian and Mackay (2000) New Engl. J. Med. 343:1020-1034; Davidson and Diamond (2001) New Engl. J. Med 345:340-350).

Methods for modulating the immune response have been used in the treatment of cancer, e.g. melanoma. These methods include treatment with either cytokines, e.g. IL-2, IL-10, IL-12, tumor necrosis factor-alpha (TNFalpha), IFNy, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF), or with cytokine antagonists (e.g. antibodies). Interleukin-10 was first characterized as a cytokine synthesis-inhibiting factor (CSIF, see e.g.

Florentino et al. (1989), J. Exp. Med. 170:2081-2095). IL-10 is a pleiotropic cytokine produced by T cells, B cells, and monocytes that can function as both an immunosuppressive and an immunostimulatory agent (see, e.g., Groux et al. (1998), J. Immunol. 160: 3188-3193 and Hagenbaugh et al (1997), J. Exp. Med. 185:2101-2110).

Animal models suggest that IL-10 can induce NK cell activation and promote the destruction of target cells in a dose-dependent manner (see, e.g., Zheng et al. (1996) J. Exp. Med.

184:579-584; Kundu et al. (1996) J. Natl. Cancer Inst. 88:536-541). Further investigations show that the presence of IL-10 in the tumor microenvironment correlates with better patient survival (see e.g. Lu et al. (2004) J. Clin. Oncol. 22:4575-4583).

Unfortunately, the serum half-life of IL-10 is relatively short, ie 2-6 hours (see, eg, Smith et al. (1996) Cellular Immunol. 173:207-214). The present invention addresses this problem by providing methods for using a modified form of IL-10, e.g. a pegylated IL-10, for the treatment of cancer. In addition to a longer half-life in serum, the pegylated form of IL-10 surprisingly showed increased tumor killing activity, e.g. via increased recruitment of CD8+ T cells to the tumor site, compared to non-pegylated IL-10.

The documents WO9519780A1, WO9703690A1 and WO0226265A2 describe uses of pegylated IL-10 (PEG-IL-10) for the treatment of cancer and the production of pegylated IL-10.

Summary of the invention

The present invention is based on the discovery that pegylated IL-10 is an improved modulator of tumor growth.

The present invention provides pegylated interleukin-10 (PEG-IL-10) for use in preventing metastases of cancer or tumor in the lungs as defined in the patent claims, comprising contacting the tumor with an effective amount of a pegylated interleukin-10 (PEG-IL-10). PEG-IL-10 can be mono-PEG-IL-10. The PEG-IL-10 in question may comprise an SC-PEG-12K linker. In an alternative embodiment, the PEG-IL-10 in question may comprise a methoxy-PEG-aldehyde (PALD-PEG) linker. The PALD-PEG linker may comprise a PEG molecule having a molecular weight selected from the group consisting of 5 KDa, 12 KDa or 20 KDa. According to the description, the PEG-IL-10 in question inhibits the growth of the tumor or cancer, or the PEG-IL-10 reduces the size of the tumor or cancer. The PEG-IL-10 in question gives increased infiltration of CD8+ T cells into the tumor compared to non-pegylated IL-10. In another embodiment, PEG-IL-10 provides increased expression of at least one inflammatory cytokine which can be selected from the group consisting of IFNγ, IL-4, IL-6, IL-10 and RANK-ligand (RANK-L). In certain embodiments, PEG-IL-10 is administered together with at least one chemotherapeutic agent. The chemotherapeutic agent can be at least one of the chemotherapeutic agents in table 16. The tumor or form of cancer is selected from the group consisting of: colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma and leukemia.

Detailed description of the invention

As used herein, including the appended claims, the singular forms of words such as "a", "an", "the" or "it" include the corresponding plural forms unless the context clearly dictates otherwise.

I. Definitions

"Activation", "stimulation" and "treatment" as applied to cells or receptors can have the same meaning, e.g. activation, stimulation or treatment of a cell or receptor with a ligand, unless otherwise indicated by the context or specifically stated. "Ligand" can be natural and synthetic ligands, e.g. cytokines, cytokine variants, cytokine analogues, cytokine muteins and binding preparations derived from antibodies. "Ligand" can be small molecules, e.g. peptide-like cytokine compounds and peptide-like antibody compounds. "Activation" can refer to cell activation regulated by internal mechanisms, as well as by extrinsic or environmental factors. The "response" to e.g. a cell, a tissue, an organ or an organism can be a change of the biochemical or physiological behaviour, e.g. concentration, density, adhesion or migration in a biological compartment, gene expression activity or differentiation state where the change correlates with activation, stimulation or treatment, or with internal mechanisms, e.g. genetic programming.

The "activity" of a molecule can describe or refer to the binding of the molecule to a ligand or to a receptor, to catalytic activity, to the ability to stimulate gene expression or cell signaling, cell differentiation or cell maturation, to antigenic activity, or to modulation of the activities of other molecules. The "activity" of a molecule can also refer to activity in terms of modulating or maintaining cell-cell interactions, e.g. adhesion, or activity in terms of preserving a cell's structure, e.g. cell membranes or cytoskeleton. "Activity" can also mean specific activity, e.g. [catalytic activity]/[mg protein], or [immunological activity]/[mg protein], or the concentration in a biological department. "Proliferative activity" can be an activity which promotes, which is necessary for or which is specifically associated with e.g. normal cell division, as well as with cancer, tumors, dysplasia, cell transformation, metastasis and angiogenesis.

"Administration" and "treatment" as applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid refer to contact of an exogenous pharmaceutical, therapeutic or diagnostic agent, an exogenous pharmaceutical, therapeutic or diagnostic compound or an exogenous pharmaceutical, therapeutic or diagnostic preparation and the animal, human, individual, cell, tissue, organ or biological fluid. "Supply" and "treatment" can e.g. refer to therapeutic methods, placebo methods, pharmacokinetic methods, diagnostic methods, research methods and experimental methods. "Treatment of a cell" can be contact between a reagent and the cell, as well as contact between a reagent and a liquid, if the liquid is in contact with the cell.

"Administration" and "treatment" also mean in vitro and ex vivo treatment, e.g. of a cell, with a reagent, a diagnostic preparation, a binding preparation or another cell. "Treatment" as applied to a human, a veterinary subject or an experimental subject refers to therapeutic treatment and prophylactic or preventive measures, to research and diagnostic applications. "Treatment" applied to a human, a veterinary medical subject or a research subject or to a cell, tissue or organ may be contact between PEG-IL-10 and a human or animal, a cell, a tissue, a physiological compartment or a physiological fluid. "Treatment of a cell" can also be situations in which PEG-IL-10 contacts the IL-10 receptor (heterodimers of IL-10R1 and IL-10R2), e.g. in liquid phase or colloidal phase, as well as situations in which an IL-10 agonist or antagonist comes into contact with a liquid, e.g. where the fluid is in contact with a cell or receptor, but where the agonist or antagonist has not been shown to contact the cell or receptor.

"Cachexia" is a wasting syndrome that includes the loss of muscle (muscle wasting) and fat and is caused by a metabolic disorder. Cachexia occurs in various forms of cancer

("cancer cachexia"), chronic obstructive pulmonary disease (COPD), advanced organ failure and AIDS. Cancer cachexia is characterized by e.g. marked weight loss, anorexia, asthenia and anemia. Anorexia is a disorder caused by a lack of motivation to eat, e.g. food aversion (see, e.g., MacDonald et al. (2003) J. Am. Coll. Surg. 197:143-161; Rubin (2003) Proc. Natl. Acad. Sci. USA 100:5384-5389; Tisdale (2002 ) Nature Reviews Cancer 2:862-871 Argiles et al (2003) Drug Discovery Today 8:838-844;

Lelli et al. (2003) J. Chemother. 15:220-225; Argiles et al. (2003) Curr. Opinion. Clin. Nutr. Metab. Care 6:401-406).

"Conservatively modified variants of PEG-IL-10" refers to both amino acid sequences and nucleic acid sequences. In the case of particular nucleic acid sequences, conservatively modified variants refer to nucleic acids that encode identical or substantially identical amino acid sequences or, if the nucleic acid does not encode an amino acid sequence, to substantially identical nucleic acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids can code for a given protein.

In the case of amino acid sequences, those skilled in the art will recognize that a single substitution in a nucleic acid, peptide, polypeptide, or protein sequence that replaces an amino acid or a small percentage of the amino acids in the encoded sequence with a conserved amino acid is a "conservatively modified variant." . Tables of conservative substitutions identify amino acids that are functionally similar to each other, as is well known in the art. An example of a conservative substitution is the replacement of an amino acid in one of the following groups with another amino acid from the same group (US Patent No. 5,767,063 assigned to Lee et al., Kyte and Doolittle (1982), J. Mol. Biol. 157: 105-132): (1) Hydrophobic: norleucine, Ile, Val, Leu, Phe, Cys and Met;

(2) Neutral hydrophilic: Cys, Ser, Thr;

(3) Acid: Asp, Glu;

(4) Basic: Asn, Gin, His, Lys, Arg;

(5) Amino acid residues affecting chain orientation : Gly, Pro; (6) Aromatic: Trp, Tyr, Phe;

(7) Small amino acids: Gly, Ala, Ser.

"Effective amount" may be an amount sufficient to alleviate or prevent a symptom or sign of the medical condition. An effective amount also means an amount sufficient to permit or promote diagnosis. An effective amount for a given patient or veterinary individual may vary depending on factors such as the condition to be treated, the general health of the patient, the procedure in terms of route of administration and dose administered, and the extent of side effects (see, e.g., US Pat. No.

5,888,530, assigned to Netti et al.). An effective amount may be the maximum dose or the maximum dosage core that avoids significant side effects or toxic effects. The effect will lead to an improvement of a diagnostic target or a diagnostic parameter by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%, where 100% is defined as the diagnostic parameter exhibited by a normal individual (see, eg, Maynard et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK). An effective amount of PEG-IL-10 will be an amount sufficient to reduce a tumor volume, inhibit tumor growth, prevent metastasis or increase CD8+ T cell infiltration at the tumor site.

"Exogenous" refers to compounds that are produced outside of an organism, cell or human body, depending on the context. "Endogenous" refers to compounds that are formed in a cell, organism or human body, depending on the context.

"Immune condition" or "immune disorder" can e.g. be a pathological inflammation, an inflammatory disorder or an autoimmune disorder or disease. "Immune condition" also refers to infections, persistent infections and proliferative conditions, e.g. cancer, tumors and angiogenesis, including infections, tumors and cancers that resist removal by the immune system. "Cancer condition" includes e.g. cancer, cancer cells, tumors, angiogenesis and precancerous conditions such as dysplasia.

"Inhibitors" and "antagonists" or "activators" and "agonists" refer to inhibitory, resp. activating molecules, e.g. for activation of e.g. a ligand, a receptor, a cofactor, a gene, a cell, a tissue or an organ. A modulator of e.g. a gene, a receptor, a ligand or a cell, is a molecule that changes an activity associated with the gene, receptor, ligand or cell, where the activity may be activated, inhibited or altered in its regulatory properties.

The modulator can act alone or use a cofactor, e.g. a protein, a metal ion or a small molecule. Inhibitors are compounds that reduce, block, prevent, delay activation, inactivate, desensitize or down-regulate e.g. a gene, a protein, a ligand, a receptor or a cell. Activators are compounds that increase, activate, promote, promote activation of, sensitize or upregulate e.g. a gene, a protein, a ligand, a receptor or a cell. An inhibitor can also be defined as a preparation that reduces, blocks or inactivates a constitutive activity. An "agonist" is a compound that interacts with a target and leads to or promotes an increased activation of the target. An "antagonist" is a compound that opposes the action of an agonist. An antagonist prevents, reduces, inhibits or neutralizes the activity of an agonist. An antagonist can also prevent, inhibit or reduce a target's constitutive activity, e.g. a target receptor, even if no agonist has been identified.

To investigate the degree of inhibition, e.g. samples or assay samples comprising a given protein or gene or a given cell or organism, with a possible activator or inhibitor and compared to control samples without the inhibitor. Control samples, i.e. samples not treated with antagonist, are assigned a relative activity value of 100%.

Inhibition is achieved if the activity value relative to the control is 90% or lower, typically 85% or lower, more typically 80% or lower, more typically 75% or lower, generally 70% or lower, more generally 65% or lower, most generally 60 % or lower, typically 55% or lower, usually 50% or lower, more commonly 45% or lower, most commonly 40% or lower, 35% or lower, 30% or lower, 25% or lower, or lower than 25% . Activation is achieved if the activity value relative to the control is approximately 110%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least twice, most often at least 2.5 times, usually at least 5 times, more commonly at least 10 times, at least 20 times, at least 40 times, or more than 40 times higher.

The endpoint in terms of activation or inhibition can be measured as follows. Activation, inhibition and response to treatment, e.g. for a cell, a physiological fluid, a tissue, an organ, an animal or a human, can be measured from an endpoint. The end point may comprise a predetermined amount or percentage of e.g. a sign of inflammation, oncogenicity or cell degranulation or secretion, e.g. release of a cytokine, toxic oxygen or a protease.

The end point can e.g. include a predetermined amount of ion flux or ion transport, cell migration, cell adhesion, cell proliferation, potential for metastasis, cell differentiation and change of phenotype, e.g. change in the expression of a gene associated with inflammation, apoptosis, transformation, cell cycle or metastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158; Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100 Timme et al (2003) Curr Drug Targets 4:251-261;

Robbins and Itzkowitz (2002) Med. Clin. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Fox. Genomics Hum. Genet.

3:101-128; Bauer et al. (2001) Glia 36:235-243; Stanimirovic and Satoh (2000) Brain Pathol. 10:113-126).

An endpoint in terms of inhibition is generally 75% of control or lower, 50% of control or lower, 25% of control or lower, or 10% of control or lower. In general, an endpoint in terms of activation is at least 150% of the control, at least twice the control, at least four times the control, or at least 10 times the control.

A "labeled" preparation can be detected, either directly or indirectly, by spectroscopic, photochemical, biochemical, immunochemical, isotope-based or chemical methods. Applicable labeling groups include e.g. <32>P, <33>P, <35>S, <14>C, <3>H, <125>I, stable isotopes, fluorescent dyes, electron dense reagents, substrates, epitope tags or enzymes, e.g. used in enzyme-linked immunoassays, or fluorettes (see, e.g., Rozinov and Nolan (1998) Chem. Biol.

5:713-728).

"Ligand" shows e.g. to a small molecule, a peptide, a polypeptide and a membrane-associated or membrane-bound molecule or a complex thereof which can act as an agonist or antagonist for a receptor. "Ligand" can also be an agent which is not an agonist or antagonist, but which can bind to the receptor without significantly affecting the receptor's biological properties, e.g. in terms of signaling or adhesion. Furthermore, "ligand" includes a membrane-bound ligand that has been altered, e.g. by chemical or recombinant methods, to a soluble version of the membrane-bound ligand. If a ligand is membrane-bound to a first cell, the receptor is usually present on a second cell. The second cell can have the same identity as the first cell or a different identity. A ligand or prescription may be fully intracellular, i.e. it may be present in the cytosol, the cell nucleus or another intracellular compartment. The ligand or receptor can change its location, e.g. from an intracellular compartment to the outer side of the plasma membrane. The complex between a ligand and a receptor is termed a "ligand-receptor complex". If a ligand and a receptor participate in a signaling pathway, the ligand is present in an upstream position and the receptor in a downstream position in the signaling pathway.

"Small molecules" are described for the treatment of physiological conditions and disorders associated with tumors and cancers. "Small molecule" is defined as a molecule with a molecular weight lower than 10 kD, typically lower than 2 kD or lower than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules comprising an inorganic component, molecules comprising a radioactive atom, synthetic molecules, peptide-like molecules and antibody-like molecules. As a therapeutic agent, a small molecule may be more permeable to cells, less prone to degradation and less able to trigger an immune response than large molecules. Small molecules, e.g. Peptide-like variants of antibodies and cytokines, as well as low molecular weight toxins, have been described (see, e.g., Casset et al. (2003) Biochem. Biophys. Res. Commun. 307:198-205; Muyldermans (2001) J. Biotechnol. 74:277-302; Li (2000) Nat. Biotechnol. 18:1251-1256; Apostolopoulos et al. (2002) Curr. Med. Chem. 9:411-420; Monfardini et al. (2002) Curr. Pharm. Dec. 8:2185-2199; Domingues et al.

(1999) Nat. Struct. Biol. 6:652-656; Sato and Sone (2003) Biochem. J. 371:603-608; US Patent No. 6,326,482, assigned to Stewart et al.).

A "chemotherapeutic agent" is a chemical compound that is useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide ("CYTOXAN"); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine, nitrogen mustards such as chlorambucil, chlornafazine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, fenesterin, prednimustine, trophosphamide and uracil mustard; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as aclacinomycins, actinomycin, authramycin, azaserin, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucin, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine and 5-FU; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostan and testolactone; antiadrenergic agents such as aminoglutethimide, mitotane and trilostane; agents providing folic acid, e.g. frolic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; the byzantine; edatraxate; Defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; the phenamine; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; "PSK"; razoxane; sizofiran; spirogermanium; tenuazoic acid; triaziquone; 2,2',2 "-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, eg paclitaxel ("TAXOL" Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel ("Taxotere", Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16) ; ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPTll; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamycins; capecitabine and pharmaceutically acceptable salts, acids or derivatives of all of the above Also included in this definition are antihormonal agents that help regulate or inhibit the action of hormones on tumors, eg antiestrogens, including eg tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifen, keoxifen, LY117018, onapristone, and toremifene (Fareston); and antiandrogenic agents such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of all of the above.

"Specific" or "selective" binding, when referring to a ligand/receptor pair, an antibody/antigen pair, or other binding pair, denotes a binding reaction that demonstrates the presence of the protein in a heterogeneous population of proteins and other biological compounds. Under specified conditions, a specified ligand thus binds to a given receptor and does not bind in significant quantities to other proteins present in the sample. The antibody or the binding preparation derived from an antibody's antigen-binding site according to the method described herein, binds to its antigen or a variant or a mutein thereof, with an affinity that is at least two times higher, at least 10 times higher, at least 20 times higher or at least 100 times higher than the affinity of any other antibody or binding preparation derived therefrom. The antibody will have an affinity that is higher than approximately 10<9 >l/mol, determined by e.g. Scatchard analysis (Munsen et al. (1980) Analyt. Biochem.

107:220-239).

"Interleukin-10" or "IL-10" as the terms are used herein, both conjugated to a polyethylene glycol and in unconjugated form, is a protein comprising two subunits that are noncovalently linked to form a homodimer. As used herein, unless otherwise indicated, "interleukin-10" and "IL10" may refer to human or mouse IL-10 (Genbank accession designations NP_000563 and M37897 or US Patent No.

6,217,857) which is also referred to as "hIL-10", respectively. "mIL-10".

"Pegylated IL-10" or "PEG-IL-10" is an IL-10 molecule with one or more polyethylene glycol molecules covalently linked to one or more than one amino acid residue in the IL-10 protein via a linker so that the connection is stable. The terms "monopegylated IL-10" and "mono-PEG-IL-10" mean that one polyethylene glycol molecule is covalently bound to a single amino acid residue in one subunit of the IL-10 dimer via a linker. The average molecular weight of the PEG group can be between approximately 5,000 and approximately 50,000 daltons. The method or site of PEG attachment to IL-10 is not critical, but the pegylation will not alter or only minimally alter the activity of the biologically active molecule. The extension of the half-life may be greater than any reduction of the biological activity. For PEG-IL-10, biological activity is typically measured by estimating the level of inflammatory cytokines (eg, TNFα, IFNγ) in the serum of individuals treated with a bacterial antigen (lipopolysaccharide, LPS) and treated with PEG-IL- 10, as described in US Patent No. 7,052,686.

As used herein, "serum half-life", abbreviated "ti/2", means the elimination half-life, i.e., the time it takes for the serum concentration of an agent to reach half of its initial or maximal value. The term "prolonged serum half-life" used herein when referring to a synthetic agent means that the synthetic agent is removed at a lower rate than either the non-synthetic, endogenous agent or the recombinantly produced version thereof.

II. Generally.

The present description describes methods for treating proliferative disorders, e.g. cancer, tumors, etc., with a pegylated-IL-10. IL-10 induces cytotoxic activity of CD8 T cells and antibody production from B cells and suppresses macrophage activity and tumor-promoting inflammation (see Chen and Zlotnik (1991) J. Immunol. 147:528-534; Groux et al. (1999 ) J. Immunol. 162:1723-1729 and Bergman et al.

(1996) J. Immunol. 157:231-238). The regulation of CD8 cells is dose-dependent, with higher doses inducing stronger cytotoxic responses, however, the applicability of recombinant hIL-10 is limited by its short half-life. PEG-IL-10 shows the unexpected ability to increase the infiltration of CD8+ T cells into a tumor, as well as an increase in the expression of inflammatory cytokines that play a role in tumor immunity. Treatment with PEG-IL-10 will therefore provide a significant improvement in tumor treatment.

III. Polyethylene glycol ("PEG")

Polyethylene glycol ("PEG") is a chemical compound that has been used to make therapeutic protein products. The verb "pegylate" means to attach at least one PEG molecule to another molecule, e.g. a therapeutic protein. For example, Adagen, a pegylated form of adenosine deaminase, is approved for the treatment of severe combined immunodeficiency disease; pegylated superoxide dismutase has undergone clinical trials for the treatment of head injury; pegylated alpha-interferon has been analyzed in phase I clinical trials for the treatment of hepatitis, while pegylated glucocerebrosidase and pegylated hemoglobin are reported to have undergone preclinical testing. The attachment of polyethylene glycol has been shown to protect against proteolysis (see, eg, Sada et al., (1991) J. Fermentation Bioengineering 71:137-139).

In its most common form, PEG is a linear or branched polyether that has hydroxyl groups at the ends and the general structure:

HO-(CH2CH2O)n-CH2CH2-OH

For linking PEG to a molecule (polypeptide, polysaccharides, polynucleotides or small organic molecules) it is necessary to activate PEG by preparing a PEG derivative with a functional group at one end or both ends. The most common reaction pathway for PEG conjugation to proteins has been to activate PEG with functional groups that are suitable for reaction with the amino group in lysine and N-terminal amino groups. More specifically, the most common reactive groups involved in linking PEG to polypeptides are the alpha-amino group or the epsilon amino group in lysine.

The reaction between a pegylation linker and a protein leads to the attachment of the PEG group, mainly in the following sites: the alpha-amino group in the N-terminus of the protein, the epsilon-amino group in the side chain of lysine residues and the imidazole group in the side chain of histidine residues. As most recombinant proteins have a single alpha amino group and a number of epsilon amino groups and imidazole groups, a number of positional isomers can be formed, depending on the linker chemistry.

Two frequently used first-generation activated monomethoxy-PEGs (mPEGs) were succinimidyl carbonate-PEG (SC-PEG, see, e.g., Zalipsky et al. (1992) Biotechnol. Appl. Biochem 15:100-114; and Miron and Wilcheck (1993 ) Bioconjug. Chem. 4:568-569) and benzotriazole carbonate-PEG (BTC-PEG, see, e.g., Dolence et al., US Patent No. 5,650,234), which preferentially reacts with lysine residues to form a carbonate bond, but which is also shown to react with histidine and tyrosine residues. The linkage to histidine residues in IFNα has been shown to be a hydrolytically unstable imidazole carbamate linkage (see, e.g., Lee and McNemar, US Patent No. 5,985,263).

Second generation PEGylation technology has been designed to avoid these unstable linkages, as well as the lack of selectivity in reactivity towards different amino acid residues. The use of a PEG-aldehyde linker provides control to a single site at the N-terminus of a polypeptide by reductive amination. IL-10 can be pegylated using different linker types and pH to obtain different forms of a pegylated molecule (see, e.g., US Patent Nos. 5,252,714, 5,643,575,

5,919,455, 5,932,462, 5,985,263 and 7,052,686).

IV. Biological activity of PEG-IL-10

Human IL-10 induces a rapid development of neutralizing antibodies when administered to immunocompetent mice. To avoid this type of neutralization, PEG-hIL-10 was administered subcutaneously to mice lacking B cells, i.e. mice unable to mount an antibody response. Well-established syngeneic tumors in these immunodeficient mice had either a significantly delayed growth or were completely rejected by PEG-hIL-10. The restriction or inhibition of tumor growth was dependent on both CD4 and CD8 T cells. After removal of the CD8 cells, the inhibitory effect of PEG-hIL-10 was completely abolished. Consequently, PEG-hIL-10 induces CD8-mediated cytotoxic responses.

Further analysis of tumor tissue showed that PEG-IL-10 produced an elevated infiltration of CD8+ T cells into the tumor at a level higher than that of non-pegylated IL-10. The level of inflammatory cytokine expression from the infiltrating CD8 cells was also higher with PEG-IL-10 treatment compared to treatment with non-pegylated IL-10. Treatment of tumor patients with PEG-IL-10 should induce a significant antitumor response and provide a significant therapeutic benefit.

An IL-10 protein described herein contains an amino acid sequence having an observed homology of at least 75%, at least 85%, or at least 90% or more, e.g. at least 95%, with a sequence from a mature IL-10 protein, i.e. a protein without leader sequences. See e.g. US Patent No. 6,217,857.

Amino acid sequence homology or sequence identity is determined by optimizing the assembly of the amino acid residues and, if necessary, introducing gaps as needed. Homologous amino acid sequences are typically intended to include natural allelic and polymorphic variation and interspecies variation in the sequence in question. Typically, homologous proteins or peptides will have from 25-100% homology (if gaps can be introduced) to 50-100% homology (if conservative substitutions are included) with the amino acid sequence of the IL-10 polypeptide. See Needleham et al., J. Mol. Biol.

48:443-453 (1970); Sankoff et al. in Time Warps, String Edits, and Macromolecules: The Theory and Practice of Sequence Comparison, 1983, Addison-Wesley, Reading, Mass.; and software packages from IntelliGenetics, Mountain View, Calif., and the University of Wisconsin Genetics Computer Group, Madison, Wis.

The IL-10 group in the PEG-IL-10 conjugates may be glycosylated or modified with non-glycosylated muteins or other analogs, including the BCRF1 (Epstein Barr virus viral IL-10) protein. Modifications in sequences coding for IL-10 can be introduced using a number of different techniques, e.g. site-directed mutagenesis [Gillman et al., Gene 8:81-97 (1979); Roberts et al., Nature 328:731-734 (1987)] and can be evaluated by a routine assay in a suitable assay of IL-10 activity. Modified IL-10 proteins, i.e. variants, may differ from the naturally occurring sequence at the primary structure level. Such modifications can be introduced by amino acid insertions, substitutions, deletions and fusions. IL-10 variants can be produced for various purposes, including prolongation of the half-life in serum, a reduction of the immune response to IL-10, simplified purification or preparation, reduced conversion of IL-10 to the monomeric subunits, improved therapeutic efficacy and a reduction of the extent and occurrence of side effects during therapeutic use.

The amino acid sequence variants are usually predetermined variants that do not occur in nature, although others may be post-translational variants, e.g. glycosylated variants. Any variant of IL-10 can be used according to the present invention as long as it has preserved a suitable level of IL-10 activity. In connection with tumors, a suitable IL-10 activity will e.g. be infiltration of CD8+ T cells in tumor sites, expression of inflammatory cytokines such as IFNγ, IL-4, IL-6, IL-10 and RANK-L from these infiltrating cells and elevated levels of TNFα or IFNγ in biological samples.

The IL-10 used in the present invention may be derived from a mammal, e.g. a human or a mouse. Human IL-10 (hIL-10) is preferred for the treatment of humans in need of IL-10 treatment. The IL-10 described here may be a recombinant IL-10. Methods that describe the preparation of human IL-10 and mouse IL-10 can be found in US patent document no.

5 231 012. Furthermore, naturally occurring or conservatively substituted variants of human IL-10 and mouse IL-10 are covered. In another embodiment of the present invention, IL-10 may be of viral origin. Cloning and expression of a viral IL-10 from Epstein Barr virus (the BCRF1 protein) is described in Moore et al., Science 248:1230 (1990).

IL-10 can be obtained in a number of different ways using standard techniques known in the art, e.g. isolated and purified from culture medium of activated cells capable of secreting the protein (eg, T cells), synthesized chemically, or synthesized by recombinant techniques (see, eg, Merrifield, Science 233:341-47 (1986); Atherton et al ., Solid Phase Peptide Synthesis, A Practical Approach, 1989, I.R.L. Press, Oxford; US Patent No. 5,231,012 which describes methods for the production of proteins with IL-10 activity, including recombinant techniques and other synthetic techniques). The IL-10 protein may be produced from nucleic acids encoding the IL-10 polypeptide using recombinant techniques. Recombinant human IL-10 is also commercially available, e.g. from Pepro Tech, Inc., Rocky Hill, N.J.

PEG-IL-10 can be prepared using techniques well known in the art. Polyethylene glycol (PEG) can be synthesized as described in e.g. Lundblad, R.L. et al. (1988) Chemical Reagents for Protein Modification CRC Press, Inc., Vol. 1, pp. 105-125. PEG can be conjugated to IL-10 using a linker as described above. In certain embodiments, the PEG-IL-10 used in the invention is a mono-PEG-IL-10 in which from 1 to 9 PEG molecules are covalently connected via a linker to the alpha-amino group in the amino acid residue at the N-terminus of one subunit in the IL-10 dimer.

IV. Therapeutic preparations, methods

PEG-IL-10 can be formulated into a pharmaceutical preparation comprising a therapeutically effective amount of IL-10 and a pharmaceutical carrier. A "therapeutically effective amount" is an amount sufficient to produce the desired therapeutic result. Such an amount preferably has minimal negative side effects. The amount of PEG-IL-10 administered in the treatment of an IL-10 treatable condition is based on the IL-10 activity of the conjugated protein, which can be determined by IL-10 activity assays known in the art. The therapeutically effective amount for a given patient in need of such treatment can be determined by taking into account various factors, e.g. the condition to be treated, the patient's general state of health, the administration method, and/or the extent of side effects. In connection with tumors, a suitable IL-10 activity will e.g. be infiltration of CD8 T cells into tumor sites, expression of inflammatory cytokines such as IFNγ, IL-4, IL-6, IL-10 and RANK-L from these infiltrating cells and an elevated level of TNFα or IFNγ in biological samples.

The therapeutically effective amount of pegylated IL-10 can vary from approximately 0.01 to approximately 100 pg of protein per kg body weight per day. The amount of pegylated IL-10 can vary from approximately 0.1 to 20 pg protein per kg body weight per day, from approximately 0.5 to 10 pg protein per kg body weight per day, or from approximately 1 to 4 pg of protein per kg body weight per day. Delivery schemes with less frequent delivery can be used when using PEG-IL-10 according to the invention, as this conjugated form has a longer duration of action than IL-10. The pegylated IL-10 is designed in a purified state and is essentially free of aggregates and other proteins. PEG-IL-10 can be administered by continuous infusion so that an amount in the range from approximately 50 to 800 pg of protein is administered per day (ie from approximately 1 to 16 pg protein per kg body weight per day of PEG-IL-10). The amount administered daily can be varied based on the measurement of side effects and the count of blood cells.

For the preparation of pharmaceutical preparations containing mono-PEG-IL-10, a therapeutically effective amount of PEG-IL-10 is mixed with a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient. The carrier or excipient may be inert. A pharmaceutical carrier may be any compatible, non-toxic compound suitable for delivery of the IL-10 compositions to a patient.

Examples of suitable carriers include normal saline, Ringer's solution, dextrose solution and Hank's solution. Non-aqueous carriers, e.g. non-volatile oils and ethyl oleate can also be used. A carrier can be 5% dextrose/saline. Carriers may contain smaller amounts of additives, e.g. compounds that provide improved isotonicity and chemical stability, e.g. buffers and preservatives, see e.g. Remington's Pharmaceutical Sciences and U.S. Pharmacopoeia:

National Formulary, Mack Publishing Company, Easton, PA (1984).

Designs of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers in the form of e.g. lyophilized powders, porridges, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990 ) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al (eds) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

Preparations described herein can be administered orally or injected into the body. Formulations for oral use may also include compounds that further protect IL-10 against proteases in the gastrointestinal tract. Injections are usually intramuscular, subcutaneous, intradermal or intravenous. Alternatively, intra-articular injection or delivery via other routes may be used under suitable circumstances.

For parenteral administration, pegylated IL-10 can be formulated into an injectable unit dosage form (solution, suspension, emulsion) in association with a pharmaceutical carrier. See e.g. Avis et al., eds., Pharmaceutical Dosage Forms:

Parenteral Medications, Dekker, N.Y. (1993); Lieberman et al., eds., Pharmaceutical Dosage Forms: Tablets, Dekker, N.Y.

(1990); and Lieberman et al., eds., Pharmaceutical Dosage Forms: Disperse Systems, Dekker, N.Y. (1990). Alternatively, preparations described herein can be introduced into the patient's body by means of an implantable or injectable drug delivery system, see e.g. Urquhart et al., Ann. Fox. Pharmacol. Toxicol. 24:199-236, (1984); Lewis, ed., Controlled Release of Pesticides and Pharmaceuticals Plenum Press, New York (1981) and US Patent Nos. 3,773,919 and 3,270,960. Pegylated IL-10 can be administered in aqueous carriers such as water, saline or buffered carriers with or without various additives and/or diluents.

An effective amount for a given patient may vary depending on factors such as the condition to be treated, the general health of the patient, the procedure in terms of route of administration and dose administered, and the extent of side effects (see, e.g., Maynard et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

Typical veterinary subjects, experimental subjects or research subjects include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs, horses and humans.

The determination of a suitable dose is carried out by the clinician, e.g. by using parameters or factors that are known or suspected in the field to affect the treatment or that have been suggested to affect the treatment. In general, the dosage begins with an amount that is somewhat lower than the optimal dose, and the dose is gradually increased after this until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic goals include diagnosis of symptoms of e.g. the inflammation or the level of inflammatory cytokines produced. A biological agent that can be used will be derived from the same species as the animal to be treated, so that the humoral response to the reagent is minimized. Methods of administration or treatment together with another therapeutic agent, e.g. a cytokine, a steroid, a chemotherapeutic agent, an antibiotic, or radiation therapy, are well known in the art (see, e.g., Hardman et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo (eds) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., PA). An effective amount of a therapeutic agent will reduce the symptoms, e.g. the tumor size or inhibition of tumor growth, typically by at least 10%, usually by at least 20%, preferably by at least 30%, more preferably by at least 40% and most preferably by at least 50%.

WE. Applications

Also described herein is PEG-IL-10 for use in the treatment of a proliferative condition or disorder, e.g. cancer of the uterus, cervix, breast, prostate, testicles, penis, gastrointestinal tract, e.g. esophagus, oropharynx, stomach, small intestine or large intestine, colon or rectum, kidney, kidney cell, urinary bladder, bone, bone marrow, skin, head or neck, skin, liver, gall bladder, heart, lung, pancreas, salivary gland, adrenal gland, thyroid gland, brain, e.g. gliomas, ganglia, the central nervous system (CNS), the peripheral nervous system (PNS) and the immune system, e.g. spleen or spleen. PEG-IL-10 is also described for use in the treatment of e.g. immunogenic tumors, non-immunogenic tumors, dormant tumors, virus-induced cancers, e.g. epithelial cell cancer, endothelial cell cancer, squamous cell carcinomas, papillomavirus, adenocarcinomas, lymphomas, carcinomas, melanomas, leukemias, myelomas, sarcomas, teratocarcinomas, chemically induced cancers, metastasis and angiogenesis. It is also described to reduce the tolerance of a tumor cell or a cancer cell antigen, e.g. by modulating the activity of a regulatory T cell (Treg) or a CD8 T cell (see e.g.

Ramirez-Montagut, et al. (2003) Oncogene 22:3180-3187; Sawaya et al. (2003) New Engl. J. Med. 349:1501-1509; Farrar et al. (1999) J. Immunol. 162:2842-2849; Le, et al. (2001) J. Immunol.

167:6765-6772; Cannistra and Niloff (1996) New Engl. J. Med. 334:1030-1038; Osborne (1998) New Engl. J. Med. 339:1609-1618; Lynch and Chapelle (2003) New Engl. J. Med. 348:919-932;

Enzinger and Mayer (2003) New Engl. J. Med. 349:2241-2252;

Forastiere, et al. (2001) New Engl. J. Med. 345:1890-1900;

Izbicki et al. (1997) New Engl. J. Med. 337:1188-1194;

Holland et al. (ed.) (1996) Cancer Medicine Encyclopedia of Cancer, 4th ed., Academic Press, San Diego, CA).

PEG-IL-10 is also described for use in the treatment of a proliferative condition, cancer, a tumor or a precancerous condition, e.g. a dysplasia, with PEG-IL-10 and at least one other therapeutic or diagnostic agent. The second therapeutic agent can e.g. be a cytokine or a cytokine antagonist, e.g. IL-12, interferon-alpha, antiepidermal growth factor receptor, doxorubicin, epirubicin, an antifolate, e.g. methotrexate or fluorouracil, irinotecan, cyclophosphamide, radiation therapy, hormone or antihormonal therapy, e.g. androgen, estrogen, antiestrogen, flutamide or diethylstilbestrol, surgical treatment, tamoxifen, ifosfamide, mitolactol, an alkylating agent, e.g. melphalan or cisplatin, etoposide, vinorelbine, vinblastine, vindesine, a glucocorticoid, a histamine receptor antagonist, an angiogenesis inhibitor, radiation, a radiosensitizer, anthracycline, vinca alkaloid, taxane, e.g. paclitaxel and docetaxel, a cell cycle inhibitor, e.g. a cyclin-dependent kinase inhibitor, a monoclonal antibody against another tumor antigen, a complex between a monoclonal antibody and a toxin, a T-cell adjuvant, a bone marrow graft or antigen-presenting cells, e.g. dendritic cell therapy. Vaccines can be provided, e.g. as soluble protein or as a nucleic acid encoding the protein (see, e.g., Le, et al., supra; Greco and Zellefsky (eds.) (2000) Radiotherapy of Prostate Cancer, Harwood Academic, Amsterdam; Shapiro and Recht (2001 ) New Engl. J. Med. 344:1997-2008; Hortobagyi (1998) New Engl. J. Med. 339:974-984; Catalona (1994) New Engl. J. Med. 331:996-1004; Naylor et al. Hadden (2003) Int. Immunopharmacol. 3:1205-1215; The Int. Adjuvant Lung Cancer Trial Collaborative Group (2004) New Engl. J. Med. 350:351-360; Slamon, et al.

(2001) New Engl. J. Med. 344:783-792; Kudelka et al. (1998) New Engl. J. Med. 338:991-992; van Netten, et al. (1996) New Engl. J. Med. 334:920-921).

PEG-IL-10 is also described for use in the treatment of extramedullary hematopoiesis (EMH) associated with cancer. EMH has been described (see, e.g., Rao, et al. (2003) Leuk. Lymphoma 44:715-718; Lane, et al. (2002) J. Cutan. Pathol.

29:608-612).

Examples

1. General procedures.

Standard procedures in molecular biology are described (Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Volume 217, Academic Press, San Diego, CA). Standard procedures also appear in Ausubel, et al. (2001) Current Protocols in Molecular Biology, Volumes 1-4, John Wiley and Sons, Inc. New York, NY, describing Cloning in Bacterial Cells and DNA Mutagenesis (Volume 1), Cloning in Mammalian Cells and Yeast (Volume 2), glycoconjugates and protein expression (volume 3) and bioinformatics (volume 4).

Procedures for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization, are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, posttranslational modification, production of fusion proteins, and glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Volume 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001 ) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). The preparation, purification and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York;

Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand/receptor interactions are available (see, eg, Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York). Methods for the production of PEG-IL-10 are described e.g. in US Patent No. 7,052,686.

Liquid flow cytometry procedures, including fluorescence-activated cell sorting (FACS), are available (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry , 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ) . Fluorescent reagents suitable for modification of nucleic acids, including nucleic acid primers and probes, polypeptides and antibodies for use as e.g. diagnostic reagents, are available (Molecular Probes (2003) Catalog, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalog, St. Louis, MO).

Standard procedures for histological analysis of the immune system are described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams and Wilkins, Phila, PA;Louis, et al.

(2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

Procedures for the treatment and diagnosis of cancer are described (see, e.g., Alison (ed.) (2001) The Cancer Handbook, Grove's Dictionaries, Inc., St. Louis, MO; Oldham (ed.) (1998) Principles of Cancer Biotherapy, 3rd ed., Kluwer Academic Publ., Hingham, MA;Thompson, et al (eds) (2001) Textbook of Melanoma, Martin Dunitz, Ltd., London, UK; Devita, et al.

(ed.) (2001) Cancer: Principles and Practice of Oncology, 6th ed., Lippincott, Phila, PA; Holland et al. (ed.) (2000) Holland-Frei Cancer Medicine, BC Decker, Phila., PA; Garrett and Sell (eds.) (1995) Cellular Cancer Markers, Humana Press, Totowa, NJ; MacKie (1996) Skin Cancer, 2nd edn, Mosby, St.

Louis; Moertel (1994) New Engl. J. Med. 330:1136-1142; Engleman (2003) Semin. Oncol. 30 (3 Suppl. 8):23—29; Mohr et al. (2003) Oncology 26:227-233).

Software packages and databases for the identification of e.g. antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence assemblies are available (see, e.g., GenBank, "Vector NTI" Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); "DeCyfer" (TimeLogic Corp., Crystal Bay, Nevada); Menne, et al. (2000) Bioinformatics 16: 741-742; Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren , et al (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690 ).

II. Pegylated IL-10

IL-10 was dialyzed against 10 mM sodium phosphate, pH 7.0, 100 mM NaCl. The dialyzed IL-10 was diluted 3.2 times to a concentration of 4 mg/ml using the dialysis buffer. Before adding the linker, SC-PEG-12K (Delmar Scientific Laboratories, Maywood, IL), 1 volume of 100 mM sodium tetraborate, pH 9.1, was added to 9 volumes of diluted IL-10 so that the pH of the IL-10 solution was raised to 8.6. The SC-PEG-12K linker was dissolved in the dialysis buffer, and an appropriate volume of the linker solution (1.8 to 3.6 mol linker per mol IL-10) was added to the diluted IL-10 solution to initiate the pegylation reaction . The reaction was carried out at 5 °C to control the reaction rate. The reaction mixture was gently stirred during the pegylation reaction. After the yield of mono-PEG-IL-10 determined by size exclusion ion HPLC (SE-HPLC) was close to 40%, the reaction was stopped by the addition of 1 M glycine solution to a final concentration of 30 mM. The pH of the reaction mixture was slowly adjusted to 7.0 using an HCl solution, and the reaction mixture was filtered through a 0.2 µm filter and stored at -80°C.

Alternatively, mono-PEG-IL-10 is prepared using methoxy-PEG-aldehyde (PALD-PEG) as a linker (Inhale Therapeutic Systems Inc., Huntsville, Alabama). PALD-PEG can have a molecular weight of 5 KDa, 12 KDa or 20 KDa. IL-10 is dialysed and diluted as described above, except that the pH of the reaction buffer is between 6.3 and 7.5. Activated PALD-PEG linker is added to the reaction buffer in a molar ratio of 1:1. Aqueous cyanoborohydride is added to the reaction mixture to a final concentration of 0.5 to 0.75 mM. The reaction is carried out at room temperature (18-25 °C) for 15-20 hours with gentle stirring. The reaction was stopped with 1 M glycine. The yield is analyzed by SE-HPLC. Mono-PEG-IL-10 is separated from unreacted IL-10, PEG-linker and di-PEG-IL-10 by gel filtration chromatography and characterized by rp-HPLC and bioassay (e.g. stimulation of IL-10 responsive cells or cell lines).

III. Tumor models

Syngeneic mouse tumor cells were injected subcutaneously or intradermally in an amount of 10% 10<5 >or 10<6 >cells per tumor inoculation. An Ep2 breast carcinoma model, CT26 colon carcinoma model, PDV6 cutaneous squamous cell carcinoma model, and 4Tl breast carcinoma model were used (see, e.g., Langowski et al. (2006) Nature 442:461-465). Immunocompetent Balb/C mice or B cell-deficient Balb/C mice were used. PEG-mIL-10 was administered to the immunocompetent mice while treatment with PEG-hIL-10 was used in the B-cell-deficient mice. The tumors now reached a size of 100-250 mm<3 >before the treatment was started. IL-10, PEG-mIL-10, PEG-hIL-10 or buffer control was administered subcutaneously in a site distant from the tumor implant. Tumor growth was typically measured twice a week using electronic sliding skin.

IV. Tumor analysis

Tumor tissue and lymph nodes were harvested at different end points for measuring the mRNA expression of a number of inflammatory markers and for performing immunohistochemical analysis of several inflammatory cell markers. The tissues were quickly frozen in liquid nitrogen and stored at -80 °C. The primary tumor growth was typically measured twice a week using an electronic caliper. The tumor volume was calculated using the formula (width<2>x length/2) where length is the longest dimension. The tumors now reached a size of 90-250 mm<3 >before the treatment was started.

V. Delivery of IL-10 and/or PEG-IL-10

mIL-10 or PEG-mIL-10 was administered to the immunocompetent mice, while PEG-hIL-10 treatment was used in the B cell-deficient mice. mIL-10, PEG-mIL-10, PEG-hIL-10 or vehicle control was administered subcutaneously in a site distant from the tumor implant. The PEG-mIL-10 used in these studies was prepared with the SC-PEG-12K linker. The biological activity of mIL-10 and PEG-mIL-10 was analyzed using a short-term proliferation bioassay using MC/9, a mouse mast cell line, which expresses endogenous mIL-10 receptors (R1 and R2). The MC/9 cells proliferate in response to simultaneous stimulation with mIL-4 and mIL-10 (the MC/9 cells do not proliferate with only mIL-4 or mIL-10). Proliferation was measured colorimetrically using Alamar Blue, a growth-indicating dye based on the detection of metabolic activity. The metabolic activity of recombinant or pegylated mIL-1 was estimated from the EC50 value, or the concentration of protein that produces half the maximal stimulation, observed in a dose-response curve (Table 1).

Table 1: MC/9 proliferation bioassay for measuring the bioactivity of the mIL-10 and PEG-mIL-10 reagents used in these studies.

Based on the MC/9 bioassay, the specific activity of the pegylated mIL-10 used in the experiments is approximately 7 times lower than the activity of the mIL-10 used (Table 1).

PEG-mIL-10 was administered every other day to mice bearing Ep2 mammary tumors. The treatment was effective in reducing tumor size and inducing tumor rejection.

Table 2: PEG-mIL-10 reduces tumor size (mm<3>) in an Ep2 breast cancer model in Balb/C mice

Treatment with PEG-mIL-10 was also effective in reducing tumor size in the PDV6, CT-26 and 4T1 syngeneic tumor models in immunocompetent mice (see Tables 3, 4 and 5).

Table 3: Study 04-M52 338: PEG-mIL-10 beginning at day 36 post-implantation reduces PDV6 tumor size (mm<3>) in C57B/6 mice

Table 4: PEG-mIL-10 beginning on day 7 post-implantation reduces tumor size of CT26 tumors (mm<3>) in BALB/cmus compared to vehicle control

Table 5: IL-10 and PEG-mIL-10 reduce tumor size (mm<3>) of 4Tl breast carcinoma

Table 6: Survey 05-M52-496. 2 weeks of treatment with mlL-10 and mPEG-IL-10 starting 19 days after implantation reduces tumor size (mm<3>) of 4Tl breast carcinoma

WE. Dose titration studies

In the dose titration, blood was collected from representative mice in each group by tail vein sampling at times corresponding to the expected peak or trough in dose levels. The harvested serum was analyzed for mIL-10 concentration using the Meso Scale Discovery platform based on a multi-array technology, a combination of electrochemiluminescence imaging and patterned arrays. A two-tailed, unpaired Student's t-test was used to compare the mean tumor volume in mIL-10 or PEG-mIL-10-treated mice grouped by serum mIL-10 concentration with the mean tumor volume in the corresponding vehicle control group. A Welch's correction was applied if two groups had different variances (p<0.05 from F-test).

Dose titration of PEG-mIL-10 and mIL-10 in 4Tl breast carcinoma-bearing mice shows that the control of primary tumor and lung metastasis can be dose titrated with both mIL-10 and PEG-mIL-10. For all given doses, PEG-mIL-10 is more effective than mIL-10 (Table 7). Treatment twice a day was started on day 17 after implantation where the average tumor volume was 84-90 mm<3>. The treatment groups consisted of 14 mice per group, while the control groups had 8 mice in each group. Tris buffer and Hepes buffer were the control for mIL-10, respectively. PEG-mIL-10.

Table 7: Survey 06-M175-1103. mIL-10 and PEG-mIL-10 reduce the primary tumor size (mm<3>) of 4Tl mammary carcinoma in BALB/c mice in a dose-dependent manner

Dose titration of PEG-mIL-10 and mIL-10 in PDV6 and mIL-10 in PDV6 squamous cell carcinoma-bearing mice shows that the control of the primary tumor can be dose titrated with both mIL-10 and PEG-mIL-10, although PEG-mIL-10 in all doses is more effective than mIL-10 (Table 8). Treatment with the highest dose of PEG-mIL-10 led to almost 100% tumor regression and subsequent resistance to re-injection of tumor cells (Table 9). Twice-daily treatment was started on day 23 after implantation when the mean tumor volume was 107-109 mm<3 >and continued until day 55 for all mIL-10-treated groups and the group treated with 0.01 mg/kg PEG-mIL- 10. The treatment with 0.1 mg/kg PEG-mIL-10 was stopped on day 48 where 100% tumor regression was observed while the remaining groups were treated until day 51. The treatment groups consisted of 10 mice per group while each vehicle control group contained 6 mice. Tris buffer and Hepes buffer were carrier controls for mIL-10, respectively. PEG-mIL-10.

Reimplantation was performed 85 days after the primary implantation and 4 weeks after the last treatment with PEG-mIL-10. There were 10 mice per group.

Table 8: Survey 06-M52-1106. mIL-10 and PEG-mIL-10 reduce tumor size (mm<3>) of PDV6 squamous cell carcinoma in C57Bl6/J mice in a dose-dependent manner.

Table 9: Survey 06-M52-1106. C57Bl/6J mice that had cleared PDV6 squamous cell carcinoma tumors after 3 weeks of treatment with PEG-mIL-10 are resistant to reimplantation in the absence of further treatment.

VII. Lung metastasis investigations

Lung metastases in the 4Tl breast carcinoma model were either quantified macroscopically after resection of the lung (Table 10) or by counting metastatic colonies in the lung after culture (Table 11) as described in Current Protocols in Immunology (section 20.2.4) John Wiley and Sons, Inc ., New York, Harlow and Lane (1999) . Briefly, lungs harvested from a 4T1 tumor-bearing mouse were dissected and treated with a mixture of collagenase and elastase followed by cultivation in a limiting dilution assay in medium containing 6-thioguanine. Only the 4Tl cells are 6-thioguanine resistant and they can be quantified by counting the number of colonies after 10-14 days of culture. Twice-daily treatment was initiated on day 17 after implantation, when the average tumor volume was 84-90 mm<3>. Tris buffer and Hepes buffer were controls for mIL-10, respectively. PEG-mIL-10. Lung metastases measured as the number of metastatic colonies cultured per lung.

Table 10: Survey 05-M52-496. 2 weeks of treatment with mlL-10 and PEG-mIL-10 starting 19 days after implantation reduces metastasis of 4Tl breast carcinoma (measured as number of lung metastases per mouse)

Table 11: Survey 06-M175-1103. mIL-10 and PEG-mIL-10 reduce lung metastases of 4Tl mammary carcinoma in BALB/c mice in a dose-dependent manner.

Administration of PEG-mIL-10 or IL-10 to 4Tl mammary carcinoma-bearing mice reduces metastasis frequency and results in increased infiltration of CD8 T cells and expression of immunostimulatory cytokines, as measured by quantitative RT-PCR (Tables 12 and 13). The number of infiltrating CD8 + T cells was counted from representative sections from several tumors that were immunohistochemically stained for the CD8 surface marker and confirmed by staining with anti-CD3 and anti-TCRαβ antibody.

Table 12: IL-10 and PEG-mIL-10 induce infiltration of CD8+ T cells in 4Tl carcinoma

PEG-mIL-10 is more effective than IL-10 for the induction of inflammatory cytokines. Total RNA from homogenized tumor samples was extracted and reverse transcribed as described previously (see, e.g., Homey et al. (2000) J. Immunol. 164:3465-3470). Complementary DNA was quantitatively analyzed for expression of cytokines by the fluorogenic 5'-nuclease PCR assay (see, e.g., Holland et al. (1991) Proc. Natl. Acad. Sci. 88:7276-7280). Specific PCR products were measured continuously using an ABI-PRISM 7700 Sequence Detection System (Applied Biosystems) over 40 cycles. The values were normalized relative to ubiquitin. Log-transformed values were analyzed by Kruskal-Wallis statistical analysis (the median method). The expression level (log-transformed) corresponds to the quantity

inflammatory cytokine expressed in the tumor sample such that the higher the expression level (log-transformed), the higher the amount of inflammatory cytokine expressed in the tumor sample.

Table 13: Administered PEG-mIL-10 induces a sustained level of inflammatory cytokines in 4Tl carcinoma 24 hours after administration of the dose

V. Removal of immune cells

CD4 + and CD8 + T cells were removed by antibody-mediated removal. 250 μg of CD4- or CD8-specific antibody was injected weekly for this purpose. Cell removal was confirmed using FACS and IHC analysis.

Removal of CD4+ T cells in B cell-deficient BALB/c mice (C.129-Igh-6<tmlcgn>) with CD4 antibodies inhibits the effect of PEG-hIL-10 on tumors (Table 14).

Table 14: PEG-hIL-10 treatment beginning 8 days after tumor implantation does not reduce tumor size (mm<3>) of CT-26 colon carcinoma after CD4 cell removal in B-cell-deficient BALB/c mice (C.129 -Igh -6<lmlCgn>).

Removal of CD8 T cells completely inhibits the effect of PEG-mIL-10 on syngeneic tumor (Table 15).

Table 15: PEG-hIL-10 treatment beginning 8 days after tumor implantation does not reduce tumor size (mm<3>) of CT-26 colon carcinoma after removal of CD8 cells in B-cell-deficient BALB/c mice

WE. Combination treatment

PEG-IL-10 is administered in combination with known chemotherapeutic agents. The chemotherapeutic agent can be administered before, simultaneously with or after the administration of PEG-IL-10. Examples of chemotherapeutic agents and dosage ranges are given in table 16.

Table 16: Dosing ranges for chemotherapeutic agents

Administration together with PEG-IL-10 may allow the use of lower and less toxic doses of the chemotherapeutic agents so that known side effects are avoided.

Claims (10)

Patent claims 1. Pegylated interleukin-10 (PEG-IL-10) for use in preventing metastases of cancer or tumor in the lungs. 2. Use of pegylated interleukin 10 (PEG_IL-10) for the production of a drug to prevent metastases of cancer or tumor in the lungs. 3. Pegylated interleukin-10 for use according to claim 1 or use according to claim 2, where the cancer or tumor is breast cancer. 4. Pegylated interleukin-10 for use according to claim 1 or use according to claim 2, wherein PEG-IL-10 comprises a methoxy-PEG-aldehyde (PALD-PEG) linker. 5. Pegylated interleukin-10 for use according to claim 1, 3 or 4 or use according to claim 2-4, where PEG-IL-10 provides increased infiltration of CD8+ T cells in the tumor compared to non-pegylated IL-10. 6. Pegylated interleukin-10 for use according to claims 1, and 3 - 5 or use according to any one of claims 2 - 5, where PEG-IL-10 increases the expression of at least one inflammatory cytokine. 7. Pegylated interleukin-10 for use according to claim 6 or use according to claim 6, where the inflammatory cytokine is selected from the group consisting of IFNγ, IL-4, IL-6, IL-10 and RANK-ligand (RANK-L). 8. Pegylated interleukin-10 for use according to any one of claims 1 and 3-7 or use according to any one of claims 2-7, where PEG-IL-10 is administered together with at least one chemotherapeutic agent. 9. Pegylated interleukin-10 for use according to claim 8 or use according to claim 8, where the chemotherapeutic agent is selected from the group consisting of Temozolomide in a dose range from 5mg - 250mg, Gemcitabine in a dose range from 200mg - lg, Doxorubicin in a dose range from lmg/m2 - 50mg/m2 and Interferon-alfa in a dose range from 1 g/kg - 300 g/kg 10. Pegylated interleukin-10 for use according to any of claims 1 and 3-9 or use according to any of claims 2-9, wherein the PEG-IL-10 is human PEG-IL-10 (PEG-hIL -10 ).